Cloning And Characterization Of Mouse PLK5, A Novel Member Of PLK Family

Polo-like kinases (PLKs) are highly conserved thronine /serine kinases playing key roles in the regulation of cell cycle progression. Up to now, four mammalian PLKs have been identified. PLK1 is an important regulatory protein in different phase of cell cycle after being activated by phosphorylation. It participates in the activation of Cyclin B/CDK1 complex in late G2/ early M phase, facilitating cells enter into M phase; assists the functional maturation and replication of centrosome and the formation of spindles; activates the formation of APC complex in mitosis telophase; facilitates normal separation and distribution of chromosome; determines whether cells can exit M phase normally and enter into cytokinesis and etc. PLK2 is expressed primarily in early G1, where it may control the entry into S phase. PLK3 appears to be expressed at constant levels throughout the cell cycle, and plays different roles in several stress response pathways, including those activated by DNA damage and spindle disruption. Meanwhile, PLK family is closely related to the migration and prognosis of tumors, boasting potential anti-cancer targeting sites. Scientists focus on studying the functions of known four PLKs as well as the discovery of new PLKs and their potential functions in cell cycle. However, there was no report about the clone of new plk gene and no record of new PLK in protein kinase images since 1997.By bioinformatics analysis and RT-PCR, we cloned an unknown gene in mouse. There is a highly homologous kinase domain in its N terminal and a PBD domain, the peculiar domain of PLK family, in its C terminal. Sequence identity analysis shows that the similarity between this new gene and PLK1 is 51% and the identity is 33%,higher than the similarity (32%) and identity (16%) between PLK4 and PLK1. Therefore, we preliminary defined it as a new member of PLK family and named it as Mouse polo-like kinase 5 (mPLK5) or mammalian PLK5. We expect further research on this new gene, such as the expression of mPLK5, the subcelluar lolization of mPLK5, the influence on the cells which overexpressed mPLK5 and so on. All of this may clarify the correlation between PLK family and cell cycle regulation, provide new potential targeting sites for the treatment of cell cycle-aberrant diseases such as cancer.Results: 1. Similar to mammalian PLK1, mouse PLK5 has two conserved domains: a highly homologous kinase domain in its N terminal and a PBD domain, the peculiar domain of PLK family, in its C terminal. Sequence identity analysis shows that the similarity between this new gene and PLK1 is 51% and the identity is 33%. Moreover, electronic PCR revealed that mouse PLK5 gene localizes to mouse chromosome 10 and consists of 1.8 kb with 15 exons. 2. Reverse transcription-polymerase chain reaction (PT-PCR) revealed that the expression of mPLK5 mRNA is restricted to the brain, eyes, liver, kidney and testis. 3. In situ hybridization of mouse brain sections with a mPLK5-specific probe indicated that PLK5 is widely expressed in difirenet brain regions, such as cerebrum, hippocampus and so on. 4. mPLK5 is mainly expressed in cerebellum, brain stem and medulla oblongata of embryonic mouse, while it is expressed in cerebrum, cerebellum, brain stem, hippocampus and medulla oblongata after born. 5. The epitopic expression of pGFP-PLK5 shows that mPLK5 was mainly located in nucleus. The mutation of conserved amino acid in Polo-box domain of mPLK5 (W417F) did not change the subcellular lolization of mPLK5. 6. Compared to control cells, overexpresion of mPLK5 may inhibite the proliferation of HeLa cells.Conclusion: We successfully cloned mPLK5, a new member of PLK family. After bioinformatics analysis, cellular and molecular experiments and confocal analysis, we found some differences among mPLK5 and other PLKs in tissue expression and the potential functions. For example, mPLK5 is specifically expressed, mainly in nerve system, kidney and testis whereas other PLKs are universally distributed. Meanwhile,Unigene analysis showed that mPLK5 is rarely expressed in tumor tissues except in testicular cancer cell lines. Our research also shows overexpresion of mPLK5 inhibited the proliferation of HeLa cells.